Generating endogenous Myh11-driven Cre mice for sex-independent gene deletion in smooth muscle cells

Abstract

Specific and efficient smooth muscle cell-targeted (SMC-targeted) gene deletion is typically achieved by pairing SMMHC-CreERT2-Tg mice with mice carrying the loxP-flanked gene. However, the transgene, CreERT2, is not controlled by the endogenous Myh11 gene promoter, and the codon-modified iCreERT2 exhibits significant tamoxifen-independent leakage. Furthermore, because the Cre-bearing bacterial artificial chromosome (BAC) is inserted onto the Y chromosome, the SMMHC-CreERT2-Tg mice strain can only exhibit gene deletions in male mice. Additionally, there is a lack of Myh11-driven constitutive Cre mice when tamoxifen usage is a concern. We used CRISPR/Cas9-mediated homologous recombination between a donor vector carrying the CreNLSP2A or CreERT2-P2A sequence and homologous arm surrounding the translation start site of the Myh11 gene to generate Cre-knockin mice. The P2A sequence enables the simultaneous translation of Cre and endogenous proteins. Using reporter mice, we assessed Cre-mediated recombination efficiency, specificity, tamoxifen-dependent controllability, and functionality in both sexes. Both constitutive (Myh11-CreNLSP2A) and inducible (Myh11-CreERT2-P2A) Cre mice demonstrated efficient, SMC-specific, sex-independent Cre recombinase activity without confounding endogenous gene expression. Combined with recently generated BAC transgenic Myh11-CreERT2-RAD mice and the Itga8-CreERT2 mouse models, our models will help expand the research toolbox, facilitating unbiased and comprehensive research in SMCs and SMC-dependent cardiovascular diseases.

Keywords: Angiogenesis; Cardiovascular disease; Vascular Biology.

Figure 1. Generation and characterization of Myh11-CreNLS^P2A KI mice.

(A) Schematic illustration of the strategy for generating endogenous Myh11-driven smooth muscle–targeted Myh11-CreNLS^P2A KI mice. (B) PCR confirmation of the Myh11-CreNLS^P2A KI insertion site for F0 founder mice and identification of Myh11-CreNLS^P2A–/–, Myh11-CreNLS^P2A+/–, and Myh11-CreNLS^P2A+/+ offspring. (C–F) Effect of Myh11-CreNLS^P2A KI on offspring Mendelian distribution (C), systolic blood pressure (D), endogenous Myh11 gene expression in the aorta, bladder, jejunum, and stomach (E) (n = 6–10 per group), and Western blot quantification of Myosin-11 (F) (coded by Myh11) protein abundance in the aorta (n = 4 per group). (G) Myh11-CreNLS^P2A KI mice were crossbred with ROSA26-driven mT/mG reporter mice. Cre activity in the aorta is proportional to the loss of tdTomato (red) signal and gain of EGFP (green) signal. Scale bars: 50 μm (upper left); 100 μm (lower right). L, aortic lumen. (H) Myh11-CreNLS^P2A KI mice were crossbred with BAF60a-floxed mice, followed by quantification of KO efficiency in the aorta and bladder using qPCR (n = 3 per group). Data are presented as mean ± SEM. Unpaired, 2-tailed Student’s t test (F and H), 1-way ANOVA (D), and 2-way ANOVA (E) followed by the Tukey test were used.

Figure 2. Comparative analysis of Cre-dependent specificity and efficiency between Myh11-CreNLS^P2A KI mice and SMMHC-CreER^T2-Tg mice.

(A) Myh11-CreNLS^P2A KI and SMMHC-CreER^T2-Tg mice were crossbred with mT/mG reporter mice. Mice were i.p. administered 50 mg/kg/day tamoxifen/corn oil for 5 consecutive days, followed by a 1 week of rest, frozen sectioning, and protein isolation from various tissues. (B) Western blot analysis of EGFP and β-actin in the aorta, bladder, and jejunum. (C) Quantification (n = 3–4 per group) of B. (D) tdTomato and EGFP signal in frozen sections of the aorta, bladder, jejunum, heart, and lower limb isolated from Myh11-CreNLS^P2A KI and SMMHC-CreER^T2-Tg mice, respectively, crossbred with mT/mG reporter mice. Data are presented as mean ± SEM. One-way ANOVA followed by the Tukey test (C). ***P < 0.001; ****P < 0.0001.

Figure 3. Characterization of tamoxifen-inducible Cre activity in Myh11-CreER^T2–P2A KI mice.

Myh11-CreER^T2–P2A KI mice were crossbred with mT/mG or LacZ reporter mice. Mice were i.p. administered 50 mg/kg/day tamoxifen/corn oil for 5 consecutive days, followed by1 week of rest, X-gal staining, frozen sectioning, or protein isolation from different tissues. (A) tdTomato and EGFP signal in frozen sections of aorta, bladder, jejunum, heart, and lower limb isolated from Myh11-CreER^{T2–P2A+/–} or Myh11-CreER^{T2–P2A–/–} mice crossbred with mT/mG reporter mice. Scale bars: 50 μm (aorta, jejunum, heart, skeletal muscle); 100 μm (bladder). L, aortic lumen. (B) Western blot analysis of CreER^T2 or EGFP and β-actin in the aorta, bladder, and jejunum, followed by quantification (n = 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (C) X-gal (β-galactosidase) staining to visualize Cre recombinase activity in the aorta, bladder, jejunum, heart, and eWAT from Myh11-CreER^{T2–P2A+/–} or Myh11-CreER^{T2–P2A–/–} mice injected with corn oil or tamoxifen. Data are presented as mean ± SEM. One-way ANOVA for CreER^T2 and 2-way ANOVA for EGFP quantification in B, followed by the Tukey test.